Compound Hb Research Articles

Diagnostic value of fetal hemoglobin Bart\u2019s for evaluation of fetal \u03b1-thalassemia syndromes: application to prenatal characterization of fetal anemia caused by undiagnosed \u03b1-hemoglobinopathy

BackgroundTo evaluate whether the quantification of fetal hemoglobin (Hb) Bart’s is useful for differentiation of α-thalassemia syndromes in the fetus and to characterize the fetal anemia associated with fetal α-hemoglobinopathy.MethodsA total of 332 fetal blood specimens collected by cordocentesis were analyzed using capillary electrophoresis and the amount of Hb Bart’s was recorded. The result was evaluated against thalassemia genotypes determined based on Hb and DNA analyses. Prenatal Hb and DNA characterization of the fetal anemia observed in two families was done.ResultsAmong 332 fetuses investigated, Hb and DNA analyses identified 152 fetuses with normal genotypes. The remaining 180 fetuses carried α-thalassemia with several genotypes. Variable amounts of Hb Bart’s were identified in all fetuses with α-thalassemia, which could be used for simple differentiation of fetal α-thalassemia genotypes. These included α+- and α0-thalassemia traits, homozygous α+-thalassemia and Hb Constant Spring (CS), Hb H disease, Hb H-CS and Hb H-Quong Sze diseases, homozygous α0-thalassemia causing the Hb Bart’s hydrops fetalis and a remain uncharacterized α-thalassemia defect. The previously undescribed interactions of Hb Queens Park and Hb Amsterdam A1 with Hb E were detected in two fetuses with Hb Bart’s of 0.5%. The Hb Queens Park-AEBart’s disease was also noted in one pregnant woman. Prenatal analysis of the fetuses with severe fetal anemia and cardiomegaly with Hb Bart’s of 9.0% and 13.6% revealed unexpectedly the homozygous Hb CS and a compound heterozygosity of Hb CS/Hb Pakse’ with Hb E heterozygote, respectively.ConclusionsThe usefulness of detecting and differentiation of fetal α-thalassemia syndromes by quantifying of Hb Bart’s was demonstrated. Apart from the fatal condition of Hb Bart’s hydrops fetalis associated with homozygous α0-thalassemia, homozygous Hb CS and a compound Hb CS/Hb Pakse’ could result in severe fetal anemia and fetal complications, prenatal diagnosis is highly recommended. The simple Hb Bart’s quantification of fetal blood should prove helpful in this matter.

Haemoglobin graphite electrodes for electrochemical energy storage

Haemoglobin (Hb) has been employed as naturally copious metal plagiaristic that attains the material with sustainable supercapacitance. Herein, a fabrication method in the course of interaction between macromolecule compound Hb with graphite has been reported over stainless steel electrode in the presence of sulphonated polysulphone (SPS) as a binder. The microstructure of fabricated Hb graphite stainless steel (SS) electrode has been ascertained through scanning electron microscopy (SEM). Electrochemical capacitance of electrodes has been examined in KOH solution (1.0 M) with reference to Ag/AgCl at a scan rate 0.001 to 0.2 Vs−1. Developed electrode has shown highest capacitance of 158.43 at scan rate of 0.001 Vs−1 along with energy and power densities of 3.86 Whkg−1 and 237.92 Whkg−1 respectively. In multiple cycles electrode rendered quite slow decrease in electrocapacitive during the first 1000 cycles (at scan rate of 0.1 Vs−1) indicating good charge–discharge with improved electrochemical cyclic stability.

Effects of Voxelotor (GBT440) Therapy on Laboratory Testing of Sickle Cell Patients

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

β-Thalassaemia and its Co-existence with Haemoglobin E and Haemoglobin S in Upper Assam Region of North Eastern India: A Hospital Based Study.

β-Thalassaemias are common genetic disorders in the Indian subcontinent and its status has not been well studied in the Upper Assam region of North Eastern India. The aim of the study was to show the prevalence of β- thalassaemias and its co-existence with Haemoglobin E and Haemoglobin S in the Upper Assam region of North Eastern India. A total of 1200 anaemic patients were investigated for β- thalassaemias. Complete Blood Count (CBC) and High Performance Liquid Chromatography (HPLC) were done for screening. Out of 1200 patients screened, 5.83% β-thalassaemia trait, 2.33% compound Hb E/β-Thalassaemia, 1.33% β-thalassaemia major and 0.42% compound Hb S/β- thalassaemia were detected. A high incidence of thalassaemia is found among the people of Upper Assam region of North Eastern India. The only way to prevent the disease is carrier detection and awareness among the people about it.

Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α‐thalassemia, 5′ δ‐globin BCL11A binding region and 3′ β‐globin enhancer

Deletions of δ- and β-globin genes are associated with different Hb F levels. To address this, we have examined hematological and molecular characteristics in a large cohort of high Hb F determinants in Thailand. A total of 160 unrelated adult subjects with heterozygous trait for high Hb F determinants and another 10 patients with compound heterozygous trait for Hb E were selectively recruited. Hematological parameters and Hb analysis were recorded, and α-thalassemia mutations were investigated. DNA deletions causing δβ(0) -thalassemia and hereditary persistence of fetal hemoglobin (HPFH) were identified using multiplex PCR and denaturing high-performance liquid chromatography (HPLC) assays developed. Four different DNA deletions were detected including the 12.6kb deletion δβ(0) -thalassemia (n=79), 79kb deletion hereditary persistence of fetal Hb (HPFH)-6 (n=65), Indian deletion-inversion (G) γ((A) γδβ)-thalassemia (n=15) and 78kb deletion Chinese (G) γ((A) γδβ)-thalassemia (n=1). Eighteen cases were found to carry α-thalassemia with 10 different genotypes. All 10 patients who had similar hematological phenotype with that of Hb E-β(0) -thalassemia were found to be compound Hb E-δβ(0) -thalassemia. Differences in hematological features as well as Hb F levels were noted and are presented comparatively. Comparison of phenotypes, genotypes, and the deletion breakpoints of these Thai high Hb F determinants indicates that differences in Hb F expression are correlated with the existence of α-thalassemia, the loss of BCL11A binding region located 5' to the δ-globin gene and the 3' β-globin enhancer, which confirms their important roles in fetal Hb expression.

Unmasking Hb Paksé (codon 142, TAA>TAT, α2) and its Combinations in Patients also Carrying Hb Constant Spring (codon 142, TAA>CAA, α2) in Northern Thailand

The incidence of Hb Paksé (codon 142, TAA>TAT, α2) might have been underestimated due to misidentifying some cases as Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) since both abnormal hemoglobins (Hbs) migrate to the same position on Hb electrophoresis or chromatography. Multiplex asymmetric allele-specific polymerase chain reaction (PCR) for identification of Hb CS and Hb Paksé, and a real-time PCR (ReTi-PCR) with SYBR Green1 high resolution melting (HRM) analysis, for detection of the α-thalassemia-1 (α-thal-1) Southeast Asian (– –SEA/) type deletion, were performed on 114 blood samples collected from subjects who lived in northern Thailand. These samples were previously identified as carrying Hb CS by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC). Five out of 114 (4.4%) samples were found to carry Hb Paksé with four different genotypes including Hb Paksé trait, compound Hb CS/Hb Paksé, Hb H-Hb Paksé disease and Hb H-Hb Paksé-Hb E disease. These results suggested that Hb Paksé and its various combinations can be misidentified as Hb CS. Although the clinical symptoms of Hb Paksé and Hb CS are similar, to prevent erroneous epidemiological data on Hb CS as well as underestimating the prevalence of Hb Paksé in northern Thailand, DNA analysis is recommended to be performed in all cases when peaks of Hb CS/Hb Paksé are detected on CE or HPLC.

Symptomatic Erythrocytosis Associated with a Compound Heterozygosity for Hb Lepore-Boston-Washington (δ87-β116) and Hb Johnstown [β109(G11)Val→Leu, GTG>TTG

Hb Johnstown [β109(G11)Val→Leu, GTG>TTG] has previously been described as a high oxygen affinity variant in a heterozygous state and in combination with β0-thalassemia (β0-thal). Because the variant does not separate from Hb A by routine methods it may be easily missed unless clinical suspicion is high. Hb Lepore-Boston-Washington (Hb LBW; δ87-β116) is a δβ hybrid variant that clinically manifests similarly to a β+-thal. Hb LBW is not detected by routine polymerase chain reaction (PCR) sequencing but is easily detected by electrophoretic methods. We describe a 19-year-old African American male with a compound heterozygosity for Hb Johnstown and Hb LBW. The patient presented with progressively worsening chest pains, headaches and erythrocytosis. He was repeatedly phlebotomized with symptomatic improvement and subsequently was confirmed to have the high oxygen affinity hemoglobin (Hb) variant. The lowest Hb and hematocrit (packed cell volume, PCV) achieved by phlebotomy was 16.1 g/dL and 0.51 L/L, respectively. Currently, he is no longer being phlebotomized, and is feeling relatively well except for minor chest pain. It is unclear to what degree the phlebotomies contributed to his subjective improvement. The combination of Hbs Johnstown and LBW has not been heretofore described, and in this case, was associated with marked symptomatic erythrocytosis. This unique combination results in a more pronounced phenotype, similar to or slightly more severe than, compound Hb Johnstown/β0-thal. This compound hemoglobinopathy will likely not be correctly classified using a single method of Hb detection and underscores the need for multiple characterization methods when indicated by the clinical picture.

Ruthenium Agostic (Phosphinoaryl)borane Complexes: Multinuclear Solid-State and Solution NMR, X-ray, and DFT Studies

The reactivity of the (o-phosphinophenyl)(amino)borane compound HB(N(i)Pr(2))C(6)H(4)(o-PPh(2)) prepared from Li(C(6)H(4))PPh(2) and HBCl(N(i)Pr(2)) toward the bis(dihydrogen) complex RuH(2)(H(2))(2)(PCy(3))(2) (1) was studied by a combination of DFT, X-ray, and multinuclear NMR techniques including solid-state NMR, a technique rarely employed in organometallic chemistry. The study showed that the complex RuH(2){HB(N(i)Pr(2))C(6)H(4)(o-PPh(2))}(PCy(3))(2) (3), isolated in excellent yield as yellow crystals and characterized by X-ray diffraction, led in solution to PCy(3) dissociation and formation of an unsaturated 16-electron complex RuH(2){HB(N(i)Pr(2))C(6)H(4)(o-PPh(2))}(PCy(3)) (4), with a hydride trans to a vacant site. In both cases, the (phosphinoaryl)(amino)borane acts as a bifunctional ligand through the phosphine moiety and a Ru-H-B interaction, thus featuring an agostic interaction.

Hemoglobin Q-Thailand related disorders: Origin, molecular, hematological and diagnostic aspects

We describe the molecular and hematological profiles of thalassemia syndromes caused by interactions of hemoglobin (Hb) Q-Thailand [α74(EF3) Asp-His] and various hemoglobinopathies found in 52 unrelated adult Thai subjects. Ten genotypes including several previously undescribed conditions were observed, which were classified into 4 groups. Group I included 26 Hb Q-Thailand heterozygotes and a homozygotous subject. Group II included subjects with Hb Q-Thailand and other α-thalassemia alleles in trans including 1 compound Hb Q-Thailand/α +-thalassemia (− α 3.7), 2 Hb Q-Thailand/Hb Constant Spring disease and 6 Hb H/Q-Thailand disease. The average levels of Hb Q-Thailand were found to be 29.8%, 82.3%, 34.7%, 49.2–49.3% and 79.4%, respectively. Both Hbs Bart's and H were observed in addition to Hb Q-Thailand in all 6 cases with Hb Q-H disease but not in a homozygous Hb Q-Thailand. Group III included 7 double heterozygotes for Hb Q-Thailand/Hb E, 3 Hb Q-Thailand/Hb E/α +-thalassemia (−α 3.7), 3 heterozygous Hb Q-Thailand/homozygous Hb E and 1 triple heterozygote for Hb Q-Thailand/Hb Constant Spring/Hb E. In this group, Hbs E (α A 2β E 2), Q-Thailand (α QT 2β A 2) and QE (α QT 2β E 2) were observed on both HPLC and capillary electrophoresis. The Hb QE, rather than Hb Q-Thailand, was detected in all 3 cases with heterozygous Hb Q-Thailand and homozygous Hb E. The remaining two cases in group 4 were double heterozygotes for Hb Q-Thailand and β 0-thalassemia in which Hb Q-Thailand, elevated Hb A 2 (α A 2δ 2), and Hb QA 2 (α QT 2δ 2) were detected. DNA analysis identified the Hb Q-Thailand mutation (α74: GAC- CAC) and the linked (− α 4.2) in all cases. Analysis of α-globin gene haplotype provided the first evidence of a single origin of this Hb variant in Thai population.

Compound heterozygote states for Hb C/Hb Malay and Hb C/Hb E in pregnancy: A molecular and hematological analysis

Hemoglobin (Hb) C (α 2β 2 6Glu-Lys) is a variant Hb found mainly in West Africa where individuals carrying both Hb C and Hb S (α 2β 2 6Glu-Val) usually have a disease similar to sickle cell disease. The Hb C molecule has reduced solubility leading to crystal formation and hemolytic anemia. We report a hitherto undescribed interaction of Hb C and Hb Malay (α 2β 2 19Asn-Ser) in a Thai individual. She was a 24-year-old pregnant woman with moderate anemia who had the following hematologic data; Hb 8.9 g/dl, Hct 30.0%, MCV 81.0 fl, MCH 24.1 pg, MCHC 29.7 g/dl, RDW 17.1% and instead of Hb crystal a marked number of target cell in peripheral blood was observed. Hb analysis revealed 22.5% Hb Malay, 64.6% Hb C and 4.5% Hb A 2. Globin gene analyses demonstrated that she carried the β C mutation (β6: GAG-AAG) in trans to the β Malay mutation (β19: AAC-AGC). Hematologic data of the patient were compared to those of the compound heterozygote for Hb C and Hb E (α 2β 2 26Glu-Lys) found in 5 other unrelated Thai pregnant women and 11 pregnant women with Hb C heterozygote with or without co-inheritance of α-thalassemia who had much lower Hb C levels and the non-pregnant women with Hb C heterozygote and a compound Hb E/Hb Malay syndrome. Different genotype–phenotype correlations observed in these Thai patients with Hb C disorders are illustrated.

Molecular and haematological characterization of compound Hb E/Hb Pyrgos and Hb E/Hb J-Bangkok in Thai patients

We describe haematological and DNA characterization of haemoglobinopathies in Thai adolescents caused by compound heterozygosities for Hb E [beta26(B8) Glu-Lys] and two other beta-globin chain variants, Hb Pyrgos [beta83(EF7) Gly-Asp] and Hb J Bangkok [beta56(D7) Gly-Asp]. Hb analysis demonstrated that although these two beta-chain variants have separated elution profiles on liquid chromatography-based Hb analysis, they have similar alkaline electrophoretic mobilities on cellulose acetate electrophoresis. Haematological data associated with these two previously undescribed conditions were compared with those of pure carriers of the variants found in other unrelated Thai individuals. beta-Globin gene haplotypes linked to these two beta-chain variants and a simple DNA testing based on multiplex allele-specific polymerized chain reaction for differential diagnosis are presented.

MOLECULAR CHARACTERIZATION OF Hb D-PUNJAB [β121(GH4)Glu→Gln] IN THAILAND

We describe the hematological and DNA characterization of Hb D-Punjab [β121(GH4)Glu→Gln] in Thailand. Nine patients from five unrelated families were studied; four patients were simple carriers of Hb D-Punjab, two were compound heterozygotes for Hb D-Punjab/β+-thalassemia;another two patients were double heterozygotes for Hb D-Punjab and α-thalassemia-2, and one patient was a compound heterozygote for Hb D-Punjab and Hb E [β26(B8)Glu→Lys]. Typical thalassemic indices with hypochromic microcytosis were observed in compound Hb D-Punjab/β+-thalassemia and Hb D-Punjab/Hb E but normal hematological profiles were observed in the remaining cases. DNA sequencing of the β-globin gene identified the G AA→C AA substitution at codon 121 causing Hb D-Punjab in all cases, and the −28 (A→G) mutation for the β+-thalassemia alleles. β-Globin gene haplotype analysis demonstrated, for the first time, that all these Asian βD-Punjab globin genes were associated with haplotype [−++−+++], previously undescribed in other populations. The finding of Hb D-Punjab in Thailand is compatible either with an independent origin of this abnormal hemoglobin or a spread of the Hb D-Punjab gene with a single origin among Asians.

Sterically demanding ferrocene-based tris(1-pyrazolyl)borate ligands

Ferrocene-based mono- and bifunctional tris(1-pyrazolyl)borates bearing either methyl or phenyl substituents at the 3-positions of their pyrazolyl rings have been obtained from FcB(NMe 2) 2 and 1,1′-fc[B(NMe 2) 2] 2 (Fc: ferrocenyl; fc: ferrocenylene) and the corresponding pyrazole derivatives via a transamination reaction. All novel ligands have been characterized as their thallium(I) complexes, and the molecular structure of FcB(pz Ph) 3Tl (pz Ph: 3-phenylpyrazolyl) has been determined by X-ray crystallography. The compound features monomeric units with tridentate [FcB(pz Ph) 3] − ligands in the crystal lattice, which is in striking contrast to the sterically less congested complex FcB(pz) 3Tl, that forms polymeric rods in the solid state. For the purpose of comparison, the single crystal X-ray structure of the ferrocene-free parent compound HB(pz) 3Tl (pz: pyrazolyl) has also been investigated, which again revealed the conventional structural motif of monomeric complexes.

The metabolism of dihydrotachysterols: Renal side chain and non-renal nuclear hydroxylations in vivo and in vitro

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT 2 and DHT 3. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT 2) was demonstrated in human plasma after administration of oral DHT 2. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT 3 in vitro showed that 25-hydroxy-DHT 3 was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT 3-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT 3. The osteosarcoma cell line metabolized 25-OH-DHT 3 in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1α,25-dihydroxy-DHT 3. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1α,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1α,25-dihydroxy-DHT is very effective in displacing radiolabelled 1α,25-dihydroxyvitamin-D 3 and is thus most likely to be the calcaemic metabolite of DHT.

Stimulation of insulin release by benzoic acid derivatives related to the non-sulphonylurea moiety of glibenclamide: Structural requirements and cellular mechanisms

A decrease in membrane permeability to K + is the first critical event occuring in pancreatic B-cells upon stimulation by hypoglycemic sulphonylureas. Compound HB 699 (4-[2-(5-chloro-2-methoxybenzamido)ethyl]benzoic acid), the non-sulphonylurea moiety of glibenclamide stimulates B-cells by the same mechanisms as glibenclamide itself. Selected derivatives of HB 699 were used to test, with isolated mouse islets, whether this property is due to the benzoic acid end of the molecule (not present in glibenclamide) or to another active site (also present in glimenclamide). Of the two halves of HB 699, p-ethylbenzoic acid, but not 5-Cl-2-methoxybenzamide, was weakly effective. Replacement of the carboxyl group of HB 699 by various non-acidic groups decreased but did not abolish the ionic and secretory effects on B-cells. Modifications of the other end of the molecule altered the efficacy in both directions. Removal of the substituents on the benzamide ring decreased the efficacy, whereas replacement of the 5-Cl-2-methoxybenzyl group by a 1,1-diphenylethyl group or a 9-fluorenylmethyl group led to substantially more active compounds. Their cellular mode of action was however not modified. It is concluded that compound HB 699 contains two active sites, both of which can trigger insulin release by decreasing K + permeability of the B-cell membrane. K channels appear to possess, not a sulphonylurea receptor, but a target site for various chemical groups. The chemical environment of the latter may also determine their efficacy by modulating their access to the channel.

The borane analogy: CH ligand orientation in arachno- and closo-butterfly clusters

The model compound HB 2H 4CH is used as a means of examining the preferred CH ligand orientation when the borane butterfly fragment HB 4H 4 + fragment accommodates CH − to give either a closo- or arachno-HB 4H 4CH cluster. The Fenske-Hall quantum chemical technique is used to explore cluster bonding first in terms of carbide protonation and then through the interaction of HB 4H 4 + and CH −. BH is formally isolobal with Fe(CO) 3 and it is shown that the fragment-ligand orbital interactions and mechanism of CH bond weakening in HFe 4(CO) 12CH are modelled successfully using HB 4H 4CH.